Insight into "nuclear-cytoplasmic shuttling" as a developmental and differentiational capability of cells in primary culture models.

Shuttling proteins are molecules that can facilitate transport through the nuclear envelope. A very large number of proteins are involved in this process that includes nuclear pore buildup, signal, receptor and enzyme proteins. There are many examples of proteins whose biological activity depends on nucleocytoplasmic transport. Very often they are largely responsible for the proper occurrence of cell division, maturation, development and differentiation. Thanks to the well mastered methods of in vitro cell culture, it is possible to trace the levels of protein expression and their distribution in cells. Advanced molecular techniques allow for precise determination of their displacement in time. Several studies are still being carried out, using primary cultures, to identify the factors that determine the maturation, development and differentiation of cells. In understanding of the detailed mechanisms controlling cell life, the key is not the level of expression of a specific protein, but its distribution in individual cellular compartments.

Genes involved in hormone metabolism and cellular response in human ovarian granulosa cells.

Endocrinal interactions are one of the most crucial regulatory mechanisms that maintain the state of homeostasis in humans. Processes such as oogenesis, folliculogenesis, menstruation and pregnancy remain under hormonal control. A key role in folliculogenesis is played by granulosa cells. Moreover, granulosa cells take part in corpus luteum formation after ovulation. Because of that, it is important to understand the ways in which the granulosa cells, associated with those processes, respond to hormonal stimulus. In the present study, a transcriptomic analysis of human granulosa cells (GCs) was carried out with the use of expression microarrays. The results were validated by RT-qPCR. The total RNA was isolated after 1st, 7th, 15th and 30th days of long-term primary cultures. The main focus of this work was placed on the genes belonging to "Response to estradiol", "Response to follicle-stimulating-hormone", "Cellular response to hormone stimulus", "Cellular hormone metabolic process" and "Hormone biosynthetic process" gene ontology groups. These groups of genes have been associated with GC hormone metabolism and cellular response to hormones. Eighty genes belonging to these groups were identified. Those that were members of more than one of the analyzed gene ontology groups, or exhibited unique expression patterns, were selected for further analysis. All of the selected genes were described, with their expression patterns detailed. In this manuscript, two gene expression patterns have been described. The first one showed large downregulation of genes in the later stages of culture, with the second one presenting upregulation of expression after day 1 of IVC. The present research was focused on six genes found to be the most important for steroidogenesis: STAR, POR, CYP11A1, ADM, GCLC, IL1B, as well as three genes of higher expression at the later stages of long-term in vitro culture: NR2F2, BMP4, COL1A1. The main goal of the presented study was to select genes involved in response to hormonal stimulus and hormone metabolism in GC long-term in vitro culture.

Transcriptomic profile of cell cycle progression genes in human ovarian granulosa cells.

The ovarian granulosa cells (GCs) that form the structure of follicle undergo substantial modification during the various stages of human folliculogenesis. These modifications include morphological changes, accompanied by differential expression of genes, encoding proteins which are mainly involved in cell growth, proliferation and differentiation. Recent data bring a new insight into the aspects of GCs' stem-like specificity and plasticity, enabling their prolonged proliferation and differentiation into other cell types. This manuscript focuses attention on emerging alterations during GC cell cycle - a series of biochemical and biophysical changes within the cell. Human GCs were collected from follicles of women set to undergo intracytoplasmic sperm injection procedure, as a part of remnant follicular fluid. The cells were primarily cultured for 30 days. Throughout this time, we observed the prominent change in cell morphology from epithelial-like to fibroblast-like, suggesting differentiation to other cell types. Additionally, at days 1, 7, 15 and 30, the RNA was isolated for molecular assays. Using Affymetrix® Human Genome U219 Array, we found 2579 human transcripts that were differentially expressed in GCs. From these genes, we extracted 582 Gene Ontology Biological Process (GO BP) Terms and 45 KEGG pathways, among which we investigated transcripts belonging to four GO BPs associated with cell proliferation: "cell cycle phase transition", "G1/S phase transition", G2/M phase transition" and "cell cycle checkpoint". Microarray results were validated by RT-qPCR. Increased expression of all the genes studied indicated that increase in GC proliferation during long-term in vitro culture is orchestrated by the up-regulation of genes related to cell cycle control. Furthermore, observed changes in cell morphology may be regulated by a presented set of genes, leading to the induction of pathways specific for stemness plasticity and transdifferentiation in vitro.

Overview of the different methods used in the primary culture of oral mucosa cells.

The culture of primary cells in vitro has enabled to gain knowledge in the field of cell biology, disease mechanisms and to offer great potential in drug testing. To date, two main techniques of isolating and culturing oral mucosal cells, the direct explant method and the enzymatic method, dominate the literature and practice. In the present study, both techniques are discussed in detail, comparing the advantages and disadvantages of the two approaches in setting up a primary culture of oral mucosal cell. The direct explant technique is well-established and has been commonly used for the past 20-30 years. Although the method of setting up the cultures did not show much variations in the methodology described by authors, the culturing conditions varied according to the aims of the projects.

Laparoscopic removal of intra-abdominal foreign body. An unusual case.

Laparoscopic retrieval of an intra-abdominal foreign body is described. This novel use of the laparoscope has not been described before.

Vasectomy in developing countries.

Vasectomy is an acceptable technique of sterilisation in affluent societies; however, cultural barriers exist in most parts of Africa. The object of this study was to establish the prevalence of vasectomies at King George V Hospital (KGH), Durban, over a 3-year period, to determine attitudes towards vasectomy, and to identify ways of improving its acceptability. This was a retrospective study. Clinical details of case files were analysed and interviews of clients who had vasectomy were conducted telephonically or through home visits. A total of 308 (88.3%) clients were contacted. The mean age at vasectomy was 35 years (range 15-51 years). The majority, 50.3%, were Indians; 21% whites, 15.6% coloured and 12.3% were Africans; 290 of the men were married with a mean duration of marriage of 10.3 years (3-25 years). Ninety-four per cent were in a monogamous relationship and all remained married to date. Sixty-four per cent had above-matriculation educational status. Most of the patients became aware of vasectomy through family planning clinics or health personnel; only 4.9% received information through the media. Fifty-three per cent (n=164) of couples had problems with previously used contraception prior to decision for vasectomy. The complication rate was 6.5% while 1% regretted having had the procedure. Only two men (0.7%) desired reversal. On direct questioning, sexual activity remained unchanged in 95.1%, and actually improved in 3.2% of the men following vasectomy. Most believed that there is not enough information on vasectomy available, especially through the media, and that with appropriate counselling vasectomy is a safe permanent contraception that should be actively promoted.